INTERNATIONAL
JOURNAL
OF SYSTEMATIC
BACTERIOLOGY,
Oct. 1988, p. 417423
0020-7713/88/040417-07$02.00/0
Copyright 0 1988, International Union of Microbiological Societies
Vol. 38, No. 4
Purine and Pyrimidine Metabolism in Mollicutes Species
MARIANN C. McELWAIN,’ D. K. F. CHANDLER,2 M. F. BARILE,2 T. F. YOUNG,’ V. V. TRYON,4
J. W. DAVIS, JR.,5 J. P. PETZEL,6 C.-J. CHANG,’ M. V.
AND J. D. POLLACK’”
Department of Medical Microbiology and Immunology, The Ohio State University, Columbus, Ohio 43210’; Division of
Bacterial Products, Center for Biologics, Evaluation and Research, Food and Drug Administration, Bethesda, Maryland
202052; Veterinary Medical Research Institute, Iowa State University, Ames, Iowa 5001 I Department of Microbiology,
University of Texas Health Sciences Center at San Antonio, San Antonio, Texas 782844;Bronx Community College of the
City University of New York, Bronx, New York 104535;Department of Microbiology, Iowa State University, Ames, Iowa
500116; Department of Plant Pathology, University of Georgia, Grifin, Georgia 30223’; and Comprehensive Cancer
Center, The Ohio State University, Columbus, Ohio 43210’
’;
We studied the purine enzyme activities in dialyzed cytoplasmic extracts from the following eight species,
representing four genera, of Mollicutes: Mycoplasma pneumoniae FHT (T = type strain) and M129,
Mycoplasma bovigenitalium PG-1lT, Mycoplasma hominis PG-21T and 1620, Mycoplasma genitalium G-37T,
Mycoplasma hyopneumoniae JT, Ureaplasma urealyticum T960T, Spiroplasma citri Mar0c-R8A2~,and Anaeroplasma intermedium 5LA. In an investigation of purine nucleoside kinase activity we also included M . hominis
13408, 10144, 13428, 1612, 1184, and Botte. All of these Mollicutes species except U . urealyticum had purine
phosphoribosyltransferase activity for adenine, hypoxanthine, and guanine; U . urealyticum had only adenine
phosphoribosyltransferase activity. All of the organisms had nucleoside phosphorylase activity which used
either ribose 1-phosphate or deoxyribose 1-phosphate and adenine, hypoxanthine, or guanine for the synthesis
of nucleosides and adenosine, deoxyadenosine, guanosine, deoxyguanosine, inosine, or deoxyinosine in the
reverse direction. All had 5’-nucleotidase activity for adenosine monophosphate, deoxyadenosine monophosphate, inosine monophosphate, or guanosine monophosphate. Only M. hominis 1620,13408,10144, and 13428,
A. intermedium, and S . citri had pyrophosphate-dependent nucleoside kinase activity. Only S. citri had
nucleoside kinase activity with adenosine triphosphate and deoxyguanosine. We studied pyrimidine enzyme
activities in all of the Mollicutes species except M . hominis and M . bovigenitalium. All of the Mollicutes species
assayed had thymidine, thymidylate, and deoxycytidine kinase and thymidine and uridine phosphorylase
activities. All of the Mycoplasma spp. had deoxycytotidine monophosphate and cytidine-deoxycytidine
deaminase activities. All of the Mycoplasma spp. and U . urealyticum lacked deoxyuridine triphosphatase
activity. U . urealyticum lacked deoxycytidine monophosphate deaminase activity, but otherwise it resembled all
of the Mycoplasma spp. A. intermedium and S . citri differed from each other and from Mycoplasma spp. and
U . urealyticum in the patterns of pyrimidine enzyme activities. Pyrophosphate-dependent nucleoside kinase
activity was the most variably detected activity. None of the Mycoplasma spp. except four of eight strains of M .
hominis had this kinase activity. Likewise, U . urealyticum did not have the pyrophosphate-dependent
nucleoside kinase activity; however, A. intermedium and S . citri did have this enzyme activity. The absence of
deoxyuridine triphosphatase activity in all Mycoplasma spp. may be related to their proposed rapid evolution
and the relative lack of conserved sequences in their 5s ribosomal ribonucleic acids.
mCi/mmol; 2’-[2,8-’H]deoxyadenosine ([2,8-’H]dADO), 28
Ci/mmol; 2’-[8-3H]deoxyguanosine ([S-’H]dGUO), 16 Ci/
mmol; 2’-[2,8-’H]deoxyadenosine 5’-monophosphate, 17 Ci/
mmol; [2,8-3H]adenosine5’-monophosphate ([2,8-’H]AMP),
17 Ci/mmol; 2’-[5-’H]deoxycytidine 5’-monophosphate (5’HIdCMP), 22 Ci/mmol; 2’-[5-’H]deoxythymidine 5’-mOnOphosphate, 70 Ci/mmol; and 2‘-[5-3H]deoxyuridine 5’-triphosphate, 11 Ci/mmol. We purchased the following
compounds from Research Products International Corp.,
Mt. Prospect, Ill.: [8-14C]adenine ([8-14C]ADE), 50 mCi/
mmol; [8-14C]adenosine ([8-14C]ADO), 47 mCi/mmol; and
[8-14C]guanosine ([8-14C]GUO), 42.8 mCi/mmol. From
Amersham Corp., Arlington Heights, Ill., we purchased
[8-14C]inosine 5’-monophosphate ([8-14C]IMP) (59 mCi/
mmol) and [2-14C]thymidine (56.6 Ci/mmol). From ICN
Pharmaceuticals Inc., Irvine, Calif., we purchased [SI4C]AMP (58 mCi/mmol) and [6-3H]uracil (40 Ci/mmol). All
enzymes and most chemicals were purchased from Sigma
Chemical Co., St. Louis, Mo.
Organisms. Mycoplasma pneumoniae FHT (T = type
strain) (passage 6 ) and M129 (passage 16), Mycoplasma
bovigenitalium PG-llT (passage 3), Mycoplasma hominis
Previous studies of the wall-less and cytochromeless Moflicutes species suggested that characterization of the metabolic pathways or patterns of these organisms might be
useful taxonomically (28, 30). We investigated Mollicutes
purine and pyrimidine metabolism because of its obvious
linkage to the synthesis of deoxyribonucleic acid (DNA) and
ribonucleic acid in these organisms, which possess some of
the smallest genomes (6, 11) and lowest guanine-plus-cytosine contents known (6, 11).In this paper we describe our
study of purine and pyrimidine metabolism in eight Mollicutes species from four genera. A comparative study of this
aspect of Mollicutes metabolism could also offer support or
hew insights into proposals concerning the phylogeny and
rapid evolution of these organisms (34, 43, 44).
MATERIALS AND METHODS
Chemicals. The following radiolabeled compounds were
purchased from Moravek Biochemicals, Brea, Calif.: [83H]guanine ([8-3H]GUA), 10 Ci/mmol; [8-14C]hypoxanthine
([8-14]HPX), 56 mCi/mmol; [8-14C]inosine ([8-14C]INO), 56
* Corresponding author.
417
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418
INT.J. SYST.BACTERIOL.
McELWAIN ET AL.
PG-21T (passage > loo), 1620 (passage 3), 13408 (passage 3),
10144 (passage 2), 13428 (passage 3), 1612 (passage 31, 1184
(passage 8), and Botte (passage > loo), Mycoplasma genitalium G-37T (passage 12), Mycoplasma hyopneumoniae JT
(= ATCC 25934T) (passage 56), Ureaplasma urealyticum
T960[CX8IT serovar VIII (passage S), and Spiroplasma citri
Mar0c-R8A2~ (= ATCC 27556T) (passage > 100) were
obtained from our stock collections. Anaeroplasma intermedium 5LA (passage > 100) was originally obtained from
I. M. Robinson, National Animal Disease Center, Ames,
Iowa.
M . hominis 1620 was isolated from the synovial fluid of a
patient with septic arthritis (2, 37) and produces joint inflammation in experimentally infected chimpanzees; strain 13408
was isolated from a patient with nongonococcal urethritis;
strain 10144 was isolated from the upper urinary tract of a
patient with a urinary tract infection; strain 13428 was
isolated from the blood of a patient with postpartum fever;
strain 1612 was isolated from normal chimpanzees; and
strain 1184 was isolated from tissue culture. M . hominis
PG-21Tand Botte were subcultured for more than 10 years in
our laboratories.
Media and growth conditions. M . pneumoniae FHT and A4.
bovigenitalium were grown in Edward-Hayflick broth containing 21 g of PPLO broth base (Difco Laboratories, Detroit, Mich.) per liter, 0.2% glucose, and 0.002% phenol red
(pH 7.6 to 7.8). The broth was supplemented with 5% (voll
vol) fresh yeast extract (lot 30003101; Flow Laboratories,
Inc., McLean, Va.), 10% (vol/vol) heat-inactivated horse
serum (lot 24P3240; GIBCO Laboratories, Grand Island,
N.Y.), and 1,000 U of penicillin G per ml. The broth used for
growth of M . hominis (all strains) was the same except that
the pH was 6.8 to 7.0, arginine (0.15%) was added, and the
concentration of fresh yeast extract was 1%.M . genitalium
and M . pneumoniae M129 were grown in Hayflick medium
(17) supplemented with 20% (vol/vol) heat-inactivated horse
serum and 0.01% phenol red. M . hyopneumoniae was grown
in Friis broth (12) containing 25% (vol/vol) acid-adjusted
swine serum (36). Mycoplasma capricolum was grown in our
modification of Edward medium (3) supplemented with 5%
(vol/vol) heat-inactivated horse serum. U. urealyticum was
grown in modified U17-B medium, harvested, and washed as
previously described (8). S . citri was grown in R, broth (4).
The strict anaerobe Anaeroplasma intermedium was grown
in Robinson MMlO medium (33), cultured, harvested, and
washed as described previously (30, 31). M . pneumoniae
(both strains) and M . genitalium were grown attached to
plastic or glass tissue culture flasks. S. citri was grown at
30°C for 3 to 4 days. M . hyopneumoniae was incubated at
37°C for 3 to 5 days with constant shaking. All other
Mollicutes species were incubated statically at 37°C for 2 to
4 days. Unless noted differently above, aerobically grown
cells were harvested and washed as previously described
(29). All washed cells were broken by hypotonic lysis or
explosive decompression in a Pam-Bomb (29). In preliminary experiments with Acholeplasma laidlawii B-PG9, Mycoplasma gallisepticum S6, and M . bovigenitalium PG-llT,
we found essentially no difference in enzyme patterns or
specific activities whether the washed cells were broken by
hypotonic lysis or explosive decompression. In our experiments, Acholeplasma laidlawii cells were always broken by
hypotonic lysis, while the other cells were generally broken
by using the Parr-Bomb. Crude lysates were examined for
thymidine kinase activity and then fractionated by centrifugation (250,000 x g , 1 to 2 h, 4°C). The supernatant (cytoplasmic) fraction was dialyzed and used for all radioactive
assays (29). In assays involving the direct staining of polyacrylamide gels with cytoplasmic and membrane fractions of
U . urealyticum, extracts were prepared as described previously (8, 32).
Assays. In cytoplasmic fractions we studied 37 enzyme
activities involved in the salvage and synthesis of purine and
pyrimidine nucleobases, ribonucleosides, deoxyribonucleosides, ribomononucleotides , and deoxyribomononucleotides. Specific radioactive assays for the purine reactions
were performed as previously described (19, 38, 39), but
with modifications. For example, the incubation time for the
assay for 5’-nucleotidase activity was reduced to 2 to 5 min
from 4 to 8 min. Briefly, 10 to 20 pmol of radioactive
substrates was mixed with cofactors in 50 mM HEPES
(N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic
acid) at
pH 7.4. In some experiments with U . urealyticum we added
2 mM p-nitrophenyl phosphate to the pyrophosphate (PPJdependent nucleoside kinase assay to reduce phosphorolysis
of the product mononucleotide by any contaminating membrane or cytoplasmic phosphatases. Dialyzed cytoplasmic
extracts containing 20 to 40 pg of protein were used to start
all reactions. Protein concentrations were determined by
using a microassay (Bio-Rad Laboratories, Richmond,
Calif.). The final volume of the reaction mixtures was 0.1 ml.
Reaction mixtures were incubated at 37°C with shaking for 4
to 16 min. Reactions were terminated by heating preparations at 90°C for 2 min. Samples (20 pl) of the heat-stopped
mixtures were spotted onto polyethylenemine-cellulose
plates (Analtech, Inc., Newark, Del.) along with nonradioactive standards (10 pg each). Radioactive substrate and
product were separated by using aqueous 1 M LiCl or with
distilled water alone. The substrate and product detected by
ultraviolet light were scraped into counting fluid and assayed
for radioactivity by scintillation counting (model LSC 7000;
Beckman Instruments, Inc., Fullerton, Calif.) with a 5 10%
counting error. Assays were considered positive when the
experimental values were 3.0 times or more greater than the
background or control values. The controls (in duplicate)
were either complete reaction mixtures with heated cytoplasmic fractions (9S°C, 10 min) or complete reaction mixtures with no cytoplasmic fraction. Also, as additional
control reactions, when there were two substrates (in the
phosphoribosyltransferase, nucleobase phosphorylase, and
nucleoside kinase assays), we omitted phosphoribosylpyrophosphate (PRPP), ribose 1-phosphate (R-1-P)-deoxyribose
1-phosphate (dR-1-P), and the phosphate donor, respectively. Data meeting the 2 3.0-fold criterion were corrected
for control values and quenching and were calculated as
disintegrations per minute per milligram of protein. Disintegrations per minute were converted to picomoles of product
by using the specific activity values supplied by the manufacturer. For purines, we estimated that we could detect 0.3
pmol of product synthesized per min per mg of protein, a
value which is at least 10-fold greater than the value which
we could previously detect (39).
The assay procedures used for pyrimidine enzyme activities have been described previously (40, 41). For the detection of cytidine deaminase activity, we substituted cytidine
for deoxycytidine (dC) in the dC deaminase assay (41). dC
kinase (EC 2.7.1.74) activity was assessed by using the
procedure of Cheng et al. (5). For pyrimidine assays, approximately 1pmol of product synthesized per min per mg of
protein could be detected.
To further study PP,-dependent nucleoside kinase activity
in U. urealyticum, cytoplasmic and membrane extract proteins (40 pg each) were electrophoresed on polyacrylamide
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VOL. 38, 1988
NUCLEIC ACID METABOLISM IN MOLLZCUTES SPECIES
gels, and an inorganic pyrophosphatase assay was performed (8). To assess orthophosphate release, the gels were
incubated for 6 min in a modified assay mixture containing
0.05 M tris(hydroxymethy1)aminomethane hydrochloride
(pH 7.8), 2 mM PP,, 2 mM MgCl,, and either 2 mM ADO or
2 mM dADO. Assays were also performed after 10 min of
preincubation in oxidized 1 mM glutathione (Sigma) in 0.05
M tris(hydroxymethy1)aminomethane hydrochloride (pH
7.8). Oxidized glutathione inhibits U . urealyticum pyrophosphatase (8), but does not inhibit mammalian nucleoside
kinase activity (9). In each series of experiments, 0.15 U of
yeast pyrophosphatase (Sigma) was included as a control.
We also tested the gels for adenosine triphosphate (ATP)dependent nucleoside kinase activity by using an assay for
adenylate kinase (14), substituting ADO for AMP, and
evaluating adenosine 5’-diphosphate formation.
RESULTS
Of the nine members of the Mollicutes shown in Table 1,
eight ( M . genitalium, M . pneumoniae M129, M . bovigenitalium, M . hyopneumoniue, M . hominis 1620, M. hominis
PG-21T, Anaeroplasma intermedium, and S . citri), like the
other Mollicutes species studied previously (19, 25, 38, 39),
had purine phosphoribosyltransferase activity for both ADE
(EC 2.4.2.7) and HPX-GUA (EC 2.4.2.8); that is, these
organisms could synthesize AMP, IMP or guanosine monophosphate (GMP) directly from their respective nucleobases
(ADE, HPX, or GUA) and PRPP (Table 1, activities 1
through 3). However, we found that U . urealyticum had
ADE phosphoribosyltransferase activity but not HPX or
GUA phosphoribosyltransferase activity.
The extracts from all of the organisms listed in Table 1had
nucleoside phosphorylase activity (EC 2.4.2.1) in the synthetic direction when either R-1-P or dR-1-P was used. In
other words, these preparations could synthesize ADO,
dADO, INO, dINO, GUO, and d G U 0 from their respective
nucleobases (ADE, HPX, and GUA) with either R-1-P or
dR-1-P (Table 1, activities 4 through 9). Conversely, the
nucleobases could be formed from their respective nucleosides (Table 1, activities 10 through 14).
Cytoplasmic 5’-nucleotidase (EC 3.1.3.5) activity for
AMP, IMP, GMP, and deoxyadenosine 5’-monophosphate
was detected in all of the cytoplasmic preparations (Table 1,
activities 20 through 23). These 5’-nucleotidase data present
a view contrary to what we have reported previously (39).
Only M. hominis 1620, Anaeroplasma intermedium, and
S . citri had nucleoside kinase activity with PP, (Table 1,
activities 15 through 19). Only S . citri had nucleoside kinase
activity with ATP and only with dGUO as the substrate
(Table 1, activity 19). The observation that M . hominis 1620
but not M . hominis PG-21T had nucleoside kinase activity
(Table 1)prompted our study of other strains of this species
(Table 2). M. hominis 1620, 13408, 10144, 13428 in low
passage (isolated from patients with septic arthritis, nongonococcal urethritis, a urinary tract infection, and postpartum
fever, respectively) had PP,-dependent nucleoside kinase
activity but not ATP-dependent nucleoside kinase activity.
M . hominis PG-21T, Botte, 1612, and 1184, strains which
were in high passage (PG-21T and Botte) or were isolated
from a normal chimpanzee (1612) or as a tissue culture
contaminant (1184), had no detectable nucleoside kinase
activity. By polyacrylamide gel electrophoresis we detected
pyrophosphatase activity in cytoplasmic but not membrane
extracts of U . urealyticum. We found neither PP,-dependent
nucleoside kinase activity nor ATP-dependent nucleoside
kinase activity in any fraction of U . urealyticum.
419
M. genitalium, M . pneumoniae M129, Anaeroplasma intermedium, U . urealyticum, and s. citri were also examined
for nine enzyme activities involved in pyrimidine deoxyribonucleotide metabolism (Table 3). All of these organisms
had thymidine phosphorylase and thymidine kinase activities and, when tested, thymidylate kinase, dC kinase, and
uridine phosphorylase activities. All of the Mycoplasma
species and strains investigated had dCMP and dC-cytidine
deaminase activities and lacked deoxyuridine triphosphatase
(dUTPase) activity. The U . urealyticum enzymatic pattern
was similar to that of the Mycoplasma spp. except that U .
urealyticum also lacked dCMP deaminase activity. The
pyrimidine deoxyribonucleotide patterns exhibited by Anaeroplasma intermedium and S . citri were different from those
of all other Mollicutes species. Anaeroplasmu intermedium
possessed all of the enzyme activities which we studied
except dC kinase. S. citri lacked all three deaminase activities. Only S. citri lacked uridine phosphorylase activity, as
McGarrity et al. have reported previously (21).
DISCUSSION
We studied in this and previous work the purine enzymatic
activities in cytoplasmic extracts of 15 Mollicutes species
(19, 39). The use of in vitro enzymatic studies for the
identification or characterization of the functional metabolic
pathways of intact cells is always perilous. A valid criticism
of this and similar studies, which has been emphasized
before (39), is that negative enzyme reactions when crude
cell extracts are used are to be viewed with great reservation. The assays may not be sufficiently sensitive or specific
to detect activity because they lack necessary reactants or
cofactors or are inappropriately incubated, because, for
example, the background or competing enzyme activity is
too high and statistically obscures low reactivity, or because
activity is lost during cell extraction. Furthermore, the rates
(specific activities) which we report in Tables 1 and 2 for
crude cytoplasmic extracts are not quantitative indications
of metabolic flow or flux, but are only qualitative indications
of the presence of in vitro enzyme activity. Nevertheless,
the results from our studies are in agreement with the
presence or absence of enzyme activities reported by other
workers who used living cells (21-24).
We found that extracts from four Acholeplasma strains,
seven Mycoplasma strains, two Spiroplasma strains, one
Ureaplasma strain, and one Anaeroplasma strain could
interconvert purine nucleobases and R-1-P or dR-1-P with
their respective ribo- or deoxyribonucleosides. With one
exception, we found that all of these preparations synthesized AMP, IMP, and GMP from PRPP and the respective
nucleobases (i.e., they had ADE, HPX, and GUA phosphoribosyltransferase activities). The one exception was U .
urealyticum, in which we detected only ADE phosphoribosyltransferase activity. In another study, in which starch
gel electrophoresis was used, ADE phosphoribosyltransferase activity in U . urealyticum T960 was not detected (28).
We believe that this contradiction of our findings reflects
differences in the techniques used. We estimate that the
procedures described in this paper are 10- to 50-fold more
sensitive than starch gel electrophoresis and our own earlier
assays (38, 39; unpublished data). The comparative metabolic data also indicate that U . urealyticum and Mycoplasma
spp. (with the exception of some strains of M . hominis) lack
detectable nucleoside kinase activity. This suggests that a
synthetic route to the purine deoxyribonucleotide precursors
of DNA in these organisms includes nucleobases, PRPP, and
cellular ribonucleotide reductase .
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4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
5
1
2
Activity
no.
ADE
HPX
GUA
ADE t
HPX t
GUA t
ADE t
HPX t
GUA t
ADO
IN0
GUO
dADO
dGUO
ADO
IN0
GUO
dADO
dGUO
AMP
IMP
GMP
dAMPh
R-1-P
R-1-P
R-1-P
dR-1-P
dR-1-P
dR-1-P
Substrate(s)'
320 (210)
470 (370)
570 (91)
820 (140)
770 (150)
820 (150)
760 (140)
820 (140)
920 (270)
820 (100)
720 (110)
470 (870)
520 (120)
630 (190)
NNNAg
NNNA
NA/NA
NNNA
NAiNA
340 (110)
450 (120)
540 (500)
330 (300)
M12Q
M. bovigenituliurn
M. hyopneumoniae
1620
M. hominis
770 (190) 440 (270) 930 (140) 1,100 (350)
870 (150) 610 (110) 1,000 (140) 1,400 (420)
1,000 (210) 850 (150) 730 (110) 910 (210)
1,000 (290) 930 (140) 1,200 (360) 480 (130)
870 (140) 1,300 (330) 1,400 (300) 630 (120)
890 (200) 850 (130) 1,100 (320) 840 (200)
1,000 (210) 740 (140) 1,000 (150) 1,000 (320)
740 (200) 940 (290) 940 (150) 740 (100)
950 (180) 1,100 (300) 1,100 (270) 640 (120)
130 (130) 840 (230) 950 (140) 1,000 (110)
130 (200) 770 (140) 830 (170) 490 (140)
160 (420) 640 (100) 850 (130) 740 (140)
200 (190) 600 (100) 940 (240) 640 (110)
130 (300) 640 (130) 860 (160) 740 (290)
NNNA
740 (100)/NA
NNNA
NA/NA
NNNA
820 (190)/NA
NNNA
NA/NA
NNNA
600 (170)/NA
NNNA
NA/NA
NNNA
930 (170)/NA
NNNA
NA/NA
NNNA
NNNA
NNNA
750 (130)/NA
820 (150) 640 (170) 980 (210) 1,000 (260)
560 (140) 980 (210) 1,000 (190) 1,100 (360)
690 (160) 830 (120) 880 (140) 1,200 (480)
670 (130) 940 (140) 720 (110) 1,100 (390)
niae
M. geniralium M: pneumo-
930
870
1,200
310
420
600
1,100
890
920
730
370
420
640
550
NNNA
NNNA
NNNA
NNNA
NNNA
870
920
730
830
M. horninis
PG-21T"
Activitv in?
~
640 (140)
450 (91)
720 (120)
1,300 (530)
1,000 (260)
950 (200)
850 (210)
1,100 (300)
970 (210)
570 (71)
830 (100)
770 (170)
300 (11)
380 (36)
1,600 (420)/NA
1,200 (300)/NA
1,900 (360)/NA
1,500 (420)/NA
1,200 (190)/NA
1,000 (190)
950 (170)
880 (110)
540 (56)
Anaeroplasma
intermedium
800 (160)
NAe
NA
1,400 (350)
1,200 (300)
1,000 (150)
1,000 (140)
930 (150)
1,000 (200)
1,300 (330)
1,500 (300)
1,400 (300)
1,200 (350)
1,400 (290)
NNNA
NNNA
NNNA
NNNA
NNNA
1,400 (320)
1,600 (420)
1,500 (300)
1,400 (350)
U.
itrealyticum
140 (27)
76(9.8)
99 (14)
57 (5.8)
110 (13)
340 (33)
75 (17)
110 (34)
380 (41)
150 (10)
NDf
72 (8.2)
78 (11)
34 (7.1)
43 (6.7)/NA
9.9 (2.1)/NA
5.0 (0.57)/NA
27 (3.5)/NA
5.3 (0.80)/80 (2.7)
23 (2.5)
0.88 (0.21)
34 (5.5)
47 (3.6)
S. citri
Reaction conditions are described in the text.
Enzyme activities are expressed as the average number of picomoles of product synthesized per minute per milligram of protein. The numbers in parentheses are standard deviations. Three different batches
of each organism were tested.
'. Qualitatively identical results (data not shown) were obtained for three different batches of M. pneumoniae FHT.
'*Only two different batches of cells were tested; the values were in close agreement.
NA, No activity detected. Our lower limit of detection was 0.3 pmol of product synthesized per min per mg of protein.
f N D , Not done.
S Data for PP,-dependent nucleoside kinaseidata for ATP-dependent nucleoside kinase.
dAMP, Deoxyadenosine 5'-monophosphate.
5'-Nucleotidases
PP,- and ATP-dependent
nucleoside kinases
Nucleoside
phosphorylases
Phosphoribosyltransferases
Enzyme class
~
TABLE 1. Purine enzyme activities of Mollicutes species
r
0
E
m
b
W
4
r
?
cl
M
P
0
N
NUCLEIC ACID METABOLISM IN MOLLICUTES SPECIES
VOL.38, 1988
TABLE 2. PPi-dependent purine nucleoside kinase activities of
cytoplasmic extracts from M . hominis strainsa
Strain
1620'
13408
10144
13428
PG-21T'
1612
1184
Botte
Activities with the following purine nucleosides:b
ADO
IN0
GUO
dADO
dGUO
740 (100)
650 (100)
720 (140)
640 (170)
N A ~
NA
NA
NA
820 (190)
920 (170)
840 (100)
700 (110)
NA
NA
NA
NA
600 (67)
620 (1 10)
690 (100)
690 (130)
NA
NA
NA
NA
930 (170)
820 (200)
890 (170)
900 (140)
NA
NA
NA
NA
750 (130)
740 (140)
930 (200)
720 (160)
NA
NA
NA
NA
a Enzyme activities are expressed as the average number of picomoles of
purine mononucleotide product synthesized per minute per milligram of
protein when we used different purine nucleosides and PP, as the substrates.
The numbers in parentheses are standard deviations. Three different batches
of each strain were tested.
Reaction conditions are described in the text.
Data for strains 1620 and PG-21T are taken from Table 1.
NA, No activity detected (<0.3 pmol of product synthesized per min per
mg of protein).
The exception to the absence of nucleoside kinase activity
in Mycoplasma spp. is M . hominis. Four strains of M .
hominis had PP,-dependent nucleoside kinase activity, and
four strains did not. To make any formal taxonomic separation within the species based on this one finding is presently
not justifiable. It should be noted that the strains exhibiting
nucleoside kinase activity were all isolated from humans
with clinical symptoms of disease. The strains in which
nucleoside kinase activity was absent were not associated
with definitive disease; i.e., they were attenuated laboratory
strains, were isolated from tissue culture, or were isolated
from a healthy chimpanzee. As phenotypic and genotypic
variations among different strains of M . hominis have also
been reported, continued study of this observation is desirable (1, 2, 7, 16).
PP,-dependent nucleoside kinase activity has been observed only in Mollicutes species; the presence of this
activity in four of the five Mollicutes genera (Acholeplasma
spp., S . citri, Anaeroplasma intermedium, and four strains
of M . hominis) may have taxonomic usefulness (19, 38).
Furthermore, the ATP-dependent nucleoside kinase of S.
citri which utilizes dGUO but not GUO as a substrate may
be useful for the identification of Spiroplasma spp., since
this activity has been found in other Spiroplasma spp. but
not in any other Mollicutes genus (unpublished data).
421
Previously we reported our failure to find 5'-nucleotidase
activity in some Mollicutes species (39). In this study, we
found 5'-nucleotidase activity in all extracts of different
Mollicutes species. We consider the 5'-nucleotidase assay to
be particularly difficult to interpret, because the product of
the reaction is a nucleoside which can be readily converted
to the nucleobase by active nucleoside phosphorylases
present in almost all crude cytoplasmic extracts of the
Mollicutes species which have been examined (13,15,19-21,
24, 25, 29; unpublished data). Therefore, 5'-nucleotidase
activity may go undetected because the reaction product is
itself consumed. The alternate procedure is to assess the
amount of mononucleotide reactant consumed. This is unreliable because the percentage consumed is very low (the
reactant is in great excess) and also because the chromotographic separation of the mononucleotide is less efficient.
Because of these reasons, we reexamined two of the four
previously negative species (39) and found that they are both
positive for 5'-nucleotidase activity. We found that Spiroplasmafloricola OBMG and M . gallisepticum S6 are both
positive for AMP and GMP nucleotidase activities (unpublished data). We did not study Acholeplasma florum or
Mycoplasma arginini or IMP and xanthosine monophosphate nucleotidase activities. We attribute the differences
between these data and the data from previous studies (39) to
technical changes. Our 5'-nucleotidase assays are now at
least 10-fold more sensitive, and the reduced incubation time
now used for these assays has permitted us to define positive
reactions with more confidence as background and control
values are relatively lower.
In this study and elsewhere (4042), we also examined the
pyrimidine deoxyribonucleotide metabolism in cytoplasmic
extracts from 14 Mollicutes species. We found thymidine
phosphorylase activity in all Mollicutes species. This observation supports the hypothesis of Neale et al. (26, 27) and
Mitchell and Finch (25) that these organisms use a salvage
synthesis pathway for thymidine nucleotides. The hydrolysis
of deoxyuridine either by uridine phosphorylase, an activity
present in all members of the Mollicutes except some
Spiroplasma strains (21), or by thymidine phosphorylase
could provide dR-1-P for the synthesis of nucleosides such
as thymidine. We believe that dC and dCMP may be sources
of dR-1-P in those organisms which possess dC deamine
activities or dCMP deaminase activities or both by first
converting dC-dCMP to deoxyuridine-deoxyuridine monophosphate, which are then deribosylated. Mollicutes species
that lack dC-dCMP deaminase activities, like S . citri and the
TABLE 3. Pyrimidine enzyme activities of Mollicutes species"
Enzyme activitiesb
Organism
Thymidine
phosphorylase
Uridine
phosphorylase
Thymidine
kinase
M . geniralium
M . pneumoniae M129
M . capricolum
Anaeroplasma
intermedium
U . urealyticum
S. citri
1.2
2.0 (0.34)
1.6 (0.3)
5.1 (7.2)
22
32
22 (3.2)
4.9
54 (14)
0.26 (0.21)
28 (5.4)
NA
dC kinase
dC
deaminase
Cytidine
deaminase
dUTPLse
0.15 (0.04)
0.05 (0.03)
1.24 (0.21)
0.07 (0.07)
0.56 (0.14) 0.09 (0.04) 6.9
9.7
0.10 (0.04) 0.22 (0.02) 3.1 (2.6) 13 (5.4)
6.5 (2.1)
0.85 (0.06) 0.22 (0.19) 2.9 (1.6)
2.3 (0.33)
NT
0.31 (0.13)
NA
NT'
NT
NT
4.1 (5.8)
N A ~
NA
NA
1.7 (0.07)
0.90 (0.50)
0.11 (0.13)
0.5 (0.2)
0.09 (0.01)
0.09 (0.01) 0.12 (0.02)
Thymidylate
kinase
dCMP
deaminase
NA
NA
39 (7.1)
NA
46 (8.0)
NA
NA
0.23 (0.05)
a Enzyme activities are expressed as the average number of nanomoles of product produced per minute per milligram of protein. The numbers in parentheses
are standard deviations. Three different batches of each organism were tested.
Reaction conditions are described in references 4 0 4 2 .
.' NT, Not tested.
NA, No activity ( ~ 0 . 0 0 1nmol of product per min per mg of protein).
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422
INT. J. SYST. BACTERIOL.
McELWAIN ET AL.
plant epiphytes Acholeplasma flurum and S . JZoricola (40),
may not utilize dC as an intercellular source for dR-1-P.
However, they may salvage dC for incorporation into DNA.
We previously suggested that it may be possible to distinguish the genera within the Mollicutes based upon the
presence or absence of enzymes involved in pyrimidine
deoxyribonucleoside metabolism (40). In this study we demonstrated that it may be possible to distinguish members of
the genera Mycoplasma and Ureaplasma from each other
and from members of other genera within the Mollicutes.
The patterns which we found in Anaeroplasma intermedium
(this study) and in the genus Acholeplasma (40) were not
distinguishable from each other, but were different from the
patterns in other genera. However, while the enzyme activity profile of S. citri was different from the profiles of
members of other genera, it was also different from the
profile which we reported for S . JZoricola (40).
We found that all 11 Mycoplasma species which we
studied and U . urealyticum lack dUTPase activity (40, 42).
The only known function of dUTPase is to prevent deoxyuridine triphosphate from being incorporated into DNA. The
incorporation of deoxyuridine triphosphate into DNA activates the error-prone uracil-DNA glycosylase base excision
repair process, and this may result in incorrect base pairing
during such repair (10, 18, 35). Further studies will be
necessary to determine what, if any, effect the lack of
dUTPase has on Mollicutes species genotypes and on the
proposed concept of the tachytelic evolution of these unusual microorganisms (41, 44).
ACKNOWLEDGMENTS
We thank R. Ross, Veterinary Medical Research Institute, Iowa
State University, Ames, and R. Donaldson, Department of Plant
Pathology, University of Georgia, Griffin, for help in preparing M .
hyopneumoniae and S . cirri cells, respectively.
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